Staphylococcus aureus is an opportunistic pathogen that causes a wide range of diseases due to ability to produce a wide variety of virulence factors. Some of these factors are regulated by agr, a quorum sensing system. Recently, a novel quorum sensing system (luxS) has been discovered. The luxS system is found in a wide variety of bacteria and it regulates virulence factors in several pathogens. S. aureus possesses a luxS homolog that is conserved in all strains. The role of luxS in the regulation of virulence in S. aureus has not yet been investigated. Our goal is to fill this gap as a prerequisite to developing therapeutic protocols for treatment of staphylococcal infections. In the proposed research, the hypothesis is that luxS system regulates the expression of virulence factors and that it communicates with the 2 main global regulators sarA and agr. The specific aims of the proposed project are: 1) Assess the role of luxS in the regulation of staphylococcal virulence factors. The luxS gene was mutated in S. aureus by insertional inactivation. Using real-time quantitative PCR (RT qPCR) and Western blotting, expression of several virulence factors will be analyzed in the luxS mutant and compared to wild type. 2) Characterize possible crosstalk between the luxS system and the agr system. Transcriptional changes in agr associated with mutation of luxS will be defined. Expression of the agr effector molecule (RNAIII) will be measured by RT qPCR. The transcription of the luxS will also be evaluated in an agr mutant to define any effect of agr on luxS expression. The phenotype of the double mutant (agr-luxS) will also be studied. 3) Define the interaction between luxS and sarA. To define the effect of the global regulator sarA on luxS, expression of luxS will be examined in a sarA mutant by RT qPCR. The expression sarA and its homologs will be examined in the luxS mutant to define the effect of luxS on these regulators. The phenotype of the double mutant (sarA-luxS) will also be studied.